弓形虫RH株热应激蛋白40基因的克隆与原核表达  

Cloning and prokaryotic expression of heat shock protein 40 gene fromToxoplasma gondii RH strain

作　　者：路静[1,2] 张念章[2] 田维鹏[3] 殷铭阳[4] 王瑞爱[1] 周东辉[2] 朱兴全[2] 翁亚彪[1] 

机构地区：[1]华南农业大学兽医学院,广东广州510642 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室甘肃省动物寄生虫病重点实验室,甘肃兰州730046 [3]东北农业大学动物医学院,黑龙江哈尔滨150030 [4]湖南农业大学动物医学院,湖南长沙410128

出　　处：《中国兽医科学》2014年第1期73-77,共5页Chinese Veterinary Science

基　　金：国家自然科学基金资助项目(31230073,31172316);甘肃省创新研究群体计划项目(1210RJIA006)

摘　　要：为研究弓形虫热应激蛋白40(TgHSP40)的生物学特性,采用RT-PCR方法从弓形虫RH株中扩增出TgHSP40基因片段,并将其克隆到原核表达载体pET-30a(+)后转化入表达菌BL21(DE3)进行IPTG诱导表达,应用Western-blot对重组蛋白的反应原性进行分析。结果显示,重组菌可表达出分子质量约46ku的重组融合蛋白,在37℃、IPTG浓度为1.0mmol/L、诱导6h的情况下表达效果最好。重组蛋白经纯化后进行的Western-blot分析证实,表达、纯化的弓形虫RH株HSP40蛋白具有良好的反应原性,与弓形虫基因Ⅰ型的RH株、基因Ⅱ型的PRU株以及基因型为ToxoDB9的TgC7株和GJS株的阳性血清均可发生特异性反应,预期可作为弓形虫病ELISA诊断方法的候选抗原。本研究结果为弓形虫病新型诊断方法的建立奠定了基础。In order to analyze the biological characteristics of the heat shock protein 40（TgHSP40） in different genotypes of Toxoplasma gondii, the target gene was amplified by RT-PCR using a pair of speci- fic primers. Then the fragments were linked into the prokaryotic expression vector pET-30a（＋） and trans- formed into Escherichia coli BL21 （DE3） strain. The recombinant protein TgHSP40 was expressed by indu- cing with IPTG and analyzed its reactinogenicity by Western-blot. In result,the recombinant protein with a molecular weight of 46 ku was expressed in E. coli by the optimized condition：inducing with 1.0 mmol/L IPTG and shaking for 6 h at 37 ~C. Western-blot analysis indicated that the TgHSP40 protein reacted with sera from mice challenged with RH, PRU and TgC7 strains and sera from pigs that challenged with GJS strain,respectively,and possessed good reactinogenicity, indicating that it could be a potential candidate antigen for developing new detection methods for toxoplasmosis.

关 键 词：弓形虫 HSP40蛋白 克隆 原核表达 反应原性 

分 类 号：S852.723[农业科学—基础兽医学]